Construction of PIAS3/EGFP eukaryotic expression plasmid and its effects on cell cycle and apoptosis of glioma cell line U251

OBJECTIVE: To construct PIAS3/EGFP(enhanced green flurescent p rotein) eukaryotic expression plasmid,and investigate the effects of exogenous PIAS3 on cell cycle and apoptosis in human glioma cell line U251.METHODS: Standard RT-PCR was used to amplify the full-length coding sequence of PIAS3.The amplified PIAS3 gene was cloned into the eukaryotic expression vector pEGFP-N1,generating pEGFP-N1-PIAS3.Human glioma cell line U251 was transfected with pEGFP-N1-PIAS3 or mock pEGFP-N1 with lipofectamine.The protein expression was observed by a fluorescence microscope.RT-PCR and Western blot were used to determine target gene expression.Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI.Flow cytometry was also used to assay the cell cycle of the transfected cells by pEGFP-N1-PIAS3 and pEGFP-N1.RESULTS: The expression of PIAS3 mRNA and protein was increased in the cells transfected by pEGFP-N1-PIAS3.The rounding and floating cells were observed by a microscope after transient transfection 48 h,while there were no changes in the mock-transfected and non-transfected cells.After transfection,the cells of apoptosis increased.The transfected cells showed an increase in the rate of apoptosis,compared with non-transfected and mock-trasfected cells(P=0.007 3).The transfected cells were blocked at S phase of cell cycle.The percentage of G2 phase in the PIAS3-transfected cells was lower than that in the mock-transfected and non-transfected cells,while the percentage of S phase was higher,and the difference was significant(P=0.025).CONCLUSION: Exogenous PIAS3 can block U251 cell cycle at the S phase,induce the apoptosis of glioma cells.